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Although many cosmetic, personal care, consumer product and raw material supplier companies have been working for decades to eliminate animal testing, in some countries regulatory authorities still require animals for product safety testing. While there are differing hurdles to acceptance of non-animal methods around the world, a common difficulty is lack of technical training. IIVS, a non-profit and world leader in the validation, training and application of non-animal test methods, has organized a group of companies to form the Industry Council for the Advancement of Regulatory Acceptance of Alternatives (ICARAA). ICARAA is a working group which provides counsel and financial support of IIVS’ mission to increase the use and adoption of in vitro methods internationally. Led by IIVS, ICARAA activities focus on educational programs that include lectures, hands-on training and data interpretation. Many of ICARAA’s activities are currently in China where there is keen interest on the part of the regulatory authorities to understand how non-animal approaches can be used to substantiate safety. This collaboration between regulatory agencies, industry and a technical institute serves as a model example of how to promote the practical acceptance of non-animal techniques and facilitate the movement away from animal testing for regulatory purposes.
Observations on the Use of the Bovine Corneal Opacity and Permeability (BCOP) Assay to Evaluate the Eye Irritation Potential of Prototype Cosmetic Formulations Containing Salicylic Acid, Glycolic Acid and Ethanol
Prototype cosmetic formulations containing 2% salicylic acid (SA) or ethanol (10% or 50%) were classified as having minimal eye irritation potential in the BCOP assay. In contrast, three formulations containing 2% SA and ethanol (10%, 15% or 30%) resulted in a classification of moderate eye irritation potential (opacity scores were higher and histopathological injury more pronounced than 100% ethanol control). The eye irritation potential of formulations containing glycolic acid (GA) is very low (EpiOcularTM ET50 = 35 to 55 minutes). Inclusion of ethanol (5%, 10% or 15%) in formulations containing 3.2 to 3.9% GA had no impact on BCOP opacity or permeability scores (each classified as having minimal eye irritation potential); however, formulations containing either 20% or 30% ethanol, 3.9% GA and 2% SA resulted in a classification of severe eye irritation potential. In both cases opacity and permeability scores were significantly higher than for the formulations containing SA and ethanol (opacity scores were also higher than for 100% ethanol and histopathological damage extended beyond the lower stroma into the endothelium). Further work is required to understand the significance of these observations but SA may potentiate the eye irritation potential of some cosmetic formulations containing ethanol (and in particular ethanol in combination with GA).
A TRPV1 expressing clone of the human SH-SY5Y neuroblastoma cell line (Figure 1) was obtained by stable transfection, using puromycin-containing selection medium. Prior to Ca2+ measurements the TRPV1-SH-SY5Y cells were cultured in 96-well plates. Acute increase in the intracellular free Ca2+ level was measured in a semi-HTS fluorescence reader (FlexStation, Molecular Devices) using Fura-2/AM. The ratio of fluorescence at 340 (Ca2+-bound Fura-2)/380 (Fura-2) nm excitatory wavelengths was registered without interruption before and during the 2 min exposure to the test compounds. The mean value (% increase of basal Ca2+ level) from triplicate wells in the 96-well plate was monitored for each concentration from each experiment. The TRPV1 antagonist capsazepine was added simultaneously with each concentration of the chemicals in three sister wells to confirm TRPV1-mediated Ca2+ influx. The intracellular Ca2+ increase induced by the specific TRPV1-agonist capsaicin was set to 100% response for each experiment and the effect of the test products was calculated as percent of the capsaicin induced response. All test compounds were diluted in KRH-buffer and the addition to the cells was performed robotically during measurements by the FlexStation reader.
Identifying the Appropriate Protocol for Testing Surfactants and Surfactant-based Formulations in the Bovine Corneal Opacity and Permeability Assay
The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo test for predicting ocular irritation. OECD Test Guideline (TG) 437 specifies that liquid and solid surfactants are tested as 10% aqueous dilutions for 10 minutes (alternate dilutions and exposure times may be conducted with scientific rationale). Guidance Document (GD) No. 160 suggests that solid and concentrated liquid surfactants may be diluted to 10% for testing, and thus surfactant solids should not be tested using the solid chemical protocol. GD No. 160 further directs that surfactant-based formulations are usually tested neat, but could be diluted with justification, imparting some confusion in identifying the most appropriate test methods. In the absence of clear guidance, we present on the testing of a few common surfactant ingredients (sodium lauryl sulfate, Triton X-100, and benzalkonium chloride), and surfactant-based liquid and solid formulations in BCOP using standard and modified dilutions and exposures to evaluate the impact of these variables. Histopathology was performed to confirm corneal changes. We found that surfactants may not exhibit dose-related effects at high concentrations, and opacity and permeability changes should be evaluated individually in a hazard assessment. Accordingly, a framework for testing surfactants and surfactant-based formulations is proposed.
3D reconstructed skin tissues provide a more realistic model for dermally applied chemicals/products, such as cosmetics, and are expected to be used to follow-up on positive results from the in vitro genotoxicity battery1. Phase 1 and 2 of the RSMN validation using EpiDermTM tissues showed good transferability, inter- and intralaboratory reproducibility2,3. In Phase 3, the number of chemicals was extended to 29 (Table 1). Results demonstrated excellent specificity and good within-laboratory reproducibility (Table 2 and 3), while sensitivity needs further investigation.
Tiered Testing Strategy Using Validated In Vitro Assays for the Assessment of Skin and Eye Corrosion/Irritation of Pharmaceutical Intermediates
The safety of workers handling solid pharmaceutical intermediates was assessed using a tiered testing strategy based on regulatory validated in vitro assays. The Top-Down approach was initiated with the in vitro skin corrosion assay (OECD TG 431) followed by the in vitro skin irritation assay (OECD TG 439) using the reconstructed human epidermis model from MatTek Corporation. Of the ten pharmaceutical intermediates tested, nine were predicted to be non-corrosive to skin and were subsequently confirmed as non-irritants. The only intermediate predicted corrosive to skin was further tested using the Corrositex® assay (OECD TG 435) and was assigned a corrosive packing group II classification. Furthermore, three intermediates predicted non-corrosive/non-irritant to skin were tested as 20% dilutions in water in the in vitro Bovine Corneal Opacity and Permeability (BCOP) assay (OECD TG 437) and they were predicted as non-irritants to the eye. Our tiered skin and eye corrosion/irritation testing strategy proved to be a very useful platform for the assessment of the potential safety risk posed to workers during the manufacturing operations used for pharmaceutical intermediates.
Testing Framework for Prediction of Ocular Irritation Using the Bovine Corneal Opacity and Permeability (BCOP) Assay and Chorioallantoic Membrane Vascular Assay (CAMVA)
In vitro ocular irritation assays, such as the Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability (BCOP) test, are routinely used by personal care products companies because they are rapid and economical to conduct, do not require the use of live animals, and provide reliable predictive data. Previous research using an extensive CAMVA and BCOP database at Kao USA Inc. has shown that ocular irritation potential for new hair shampoos, ethanol-based hair stylers, skin cleansers, and skin lotions can be reliably predicted using a decision tree that systematically compares the ingredient composition, particularly ethanol and surfactant content, of the new formulation to previously tested formulations. Because the studies comprising this original database were conducted at a single contract laboratory, a follow-up study using a second contract laboratory was conducted to demonstrate inter-laboratory reliability of the CAMVA/BCOP data-derived decision tree for prediction of ocular irritation potential. Thirty-five personal care products were tested using the CAMVA and/or BCOP assays. The ethanol and surfactant content of each test material was evaluated, and the results of the assays were compared to the decision tree-based predictions of ocular irritation potential. Our data confirmed the ocular irritation predictions made using the decision tree model for 33 of 37 test samples (89% correlation rate) and verified the inter-laboratory reliability of the CAMVA and BCOP assays when conducted using appropriate controls. Our results also strengthened the ocular irritation decision tree model by confirming that deodorants are consistently predicted not to be ocular irritants based on composition.
In Vitro Safety Profile of Personal Care Products - Use of an In Vitro Testing Platform Based on a Reconstructed Vaginal Tissue Model
One of the common goals of industry is to confirm the safety of their products. Ethical concerns have led to the use of alternative testing methods in lieu of traditional testing methods. Several studies have shown good correlation between alternative test methods, traditional testing methods and human exposure. In the current study, the safety profile of three products with potential for vaginal exposure was assessed using the reconstructed human vaginal EpiVaginal™ model (MatTek Corporation, USA); the assay negative control (sterile, deionized water) and positive control (1% Triton® -X-100) were tested alongside. To increase the confidence in the test outcome, histopathology evaluation was conducted to assess the extent of cellular damage. Two liquid products were directly applied to the EpiVaginal™ tissues, while the wet wipe product was placed in direct contact with the tissue. Vaginal irritation expressed at ET50 values (3.32 and 12.71 hours) showed a higher irritation potential for the liquid formulations compared to the wipes (>24 hours). The lower irritation potential of the wipe product may be related to the availability of a rather limited amount of the liquid formulation in the wipes compared to the liquid formulations. Histology evaluations showed good correlation between the ET50 values and change in tissue structure. The results of this in vitro test methodology confirmed the safety profile of the products, should vaginal exposure occur during use. This two-endpoint testing platform (viability and histology) provided not only a correlative interpretation of the data, but also indication of the structural changes of the tissues exposed to the test article that are relevant to human exposure. Future plans include further exploring the capability of this in vitro testing platform for screening products before entering clinical trials.
Investigations on Reducing Ocular Irritation Associated with Harsh Ingredients by Altering Physicochemical Properties of the Formulation
With the upcoming implementation of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) in various forms throughout the world and a global movement towards a reduction in animal testing, more emphasis is placed on utilizing the inherent hazards of chemicals for classification and labeling; however, the assessment of the toxicity of chemical mixtures, particularly ocular irritation, can be complex. The ability to formulate mixtures to be less irritating with minor modifications to the physical form would be very beneficial. The present study used the Bovine Cornea Opacity and Permeability (BCOP) assay, which is an OECD-approved in vitro method to assess ocular irritation, to investigate how physical properties (e.g., viscosity) affect ocular irritation. We found that the BCOP in vitro scores of irritating chemicals from several classes, including strong bases, were diminished by altering the viscosity of the aqueous medium or by dosing the solution as a foam. Our data show that a 1% NaOH solution in water produced an in vitro score of 165; however, when the medium was thickened with 1% Carbopol®, the in vitro score dropped to 67.6. A change of this nature is significant, and if this were an EPA-registered antimicrobial cleaner, for example, this reduction in the BCOP score would lower the hazard category. Product form and usage can clearly impact exposure, and the present results suggest that modifications to the physical properties of chemical mixtures can alter their ocular irritation potential; perhaps by affecting exposure to the eye. Although no formal comparisons were performed in animals, the BCOP assay is an OECD-validated method to assess ocular irritation, and studies have shown that the BCOP assay does not under-predict the results of traditional animal tests; thus, there are no obvious reasons to suggest that the present results would not correlate to animals or humans.
Background The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo test used to evaluate ocular irritation. According to the OECD Test Guideline (TG) 437, the BCOP assay can be used to identify chemicals which induce severe/corrosive eye irritation and those that do not require classification. However, BCOP has historically under-predicted certain anionic surfactants, when tested according to the standard liquid protocol. TG 437 specifies that liquid and solid surfactants may be tested as 10% aqueous dilutions for 10 minutes (although alternate dilutions and exposure times may be conducted with scientific rationale). The relevant guidance document (GD) No. 160 suggests that solid and concentrated liquid surfactants may be diluted to 10% for testing. However, GD No. 160 further directs that surfactant-based formulations are usually tested neat, but could be diluted with justification, imparting some confusion in identifying the most appropriate test methods. Additionally, as part of the EPA classification of ocular irritation, the BCOP assay may be used to assess anti-microbial products with cleaning claims. Such products may contain surfactants and are generally tested neat for classification purposes.
Methods Since neither the basis for selecting the appropriate surfactant test methods, nor the justification for modifications are clearly presented in TG 437 or GD No. 160, we present on the testing of a few common surfactant ingredients, including sodium lauryl sulfate (SLS), Triton X-100, and benzalkonium chloride, and surfactant based formulations in the BCOP assay using standard and modified dilutions and exposures to elucidate the impact of these variables on eye irritation prediction.
Results and Discussion As examples, in vitro scores of 20.7, 28.4, and 28.3 were obtained when testing SLS at concentrations of 50, 20, and 10% for 10 minutes, showing that irritation responses were not fully concentration-dependent. As a complement to the BCOP assay, histopathology was performed to assess the surfactant-induced corneal changes. Based upon these results, a framework for testing surfactant ingredients and surfactant-based formulations is proposed.